Skin tissue wound healing mimicry material

Abstract

To provide a skin tissue wound adhesion imitation material by which, on an injury part, a tissue which imitates a bulbil is produced.SOLUTION: There is provided a skin tissue wound adhesion imitation material including, cells and gel of collagen. Average concentration of the collagen in the skin tissue wound adhesion imitation material, is 2 mg / mL or greater and 10 mg / mL or smaller, and in the skin tissue wound adhesion imitation material, (the average concentration of the collagen [mg / mL]×2)+log(the number of cells per unit volume [cells / mL]) may be, 9.3 or greater and 29.0 or smaller. In the skin tissue wound adhesion imitation material, there may be fibroblasts in the cells. The skin tissue wound adhesion imitation material includes multiple areas where the collagen concentration is different, and concentration of the collagen on at least some of the multiple areas may be 0.1 mg / mL or greater and 0.5 mg / mL or smaller, and concentration of the collagen on at least other some areas of the multiple areas may be, 2 mg / mL or greater and 8 mg / mL or smaller.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] The present invention relates to cell engineering and relates to a skin tissue wound healing mimetic material.

Background Art

[0002] A wound is healed through four complex processes: a bleeding and coagulation stage, an inflammatory stage, a proliferative stage, and a maturation stage. In general wound treatment, it is considered most important to appropriately perform epithelialization and healing that occur from the inflammatory stage to the proliferative stage.

[0003] As a conventional model for evaluating the effectiveness of wound healing by a contract research organization or the like, there is a scratch assay by monolayer cell culture. In the scratch assay, after monolayer culture is performed so that cells become confluent on a culture substrate, a part of the surface of the monolayer composed of cells is scratched to create a region where cells are detached (a pseudo-wound site), and the coverage of cells that occurs at the wound site during subsequent culture is evaluated. However, in the scratch assay, only epithelialization due to cell migration is evaluated, and the healing of the wound site in wound healing cannot be evaluated.

[0004] Non-Patent Documents 1 and 2 report that a culture substrate in which a part of collagen seeded with cells on the surface is hollowed out and the hollowed-out part is filled with collagen and fibrin not containing cells was prepared as a wound healing model. Non-Patent Documents 1 and 2 report a model that reproduces the coverage of the wound bed by cells from cell migration during culture. However, this model only evaluates cell migration and cannot evaluate the healing of the wound site in wound healing. Also, in an actual wound, granulation fills the wound site by cells producing collagen, but in this model, it is transplanted into an artificially hollowed-out part of collagen, and granulation cannot be reproduced.

[0005] Patent Document 1 reports the creation of an artificial three-dimensional model coated with collagen containing dermal cells, conforming to the shape of the human body, such as fingers and face. Furthermore, Patent Document 1 reports that, as a wound repair model, a defect was created, and then a collagen sheet was transplanted to the wound, after which cell migration, recovery of strength, and wound repair were confirmed. However, in actual wounds, granulation tissue fills the wound due to collagen production by cells, whereas in this model, collagen is artificially transplanted, and granulation tissue cannot be reproduced. In addition, since the wound is artificially covered by the collagen sheet transplant, spontaneous wound closure cannot be reproduced. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2021-078494 [Non-patent literature]

[0007] [Non-Patent Document 1] Zhuo J. Chen, "A Novel Three-Dimensional Wound Healing Model," J. Dev. Biol. 2014, 2, 198-209; doi:10.3390 / jdb2040198 [Non-Patent Document 2] Kritika Iyer, "Keratinocyte Migration in a Three-Dimensional In Vitro Wound Healing Model Co-Cultured with Fibroblasts," Tissue Eng Regen Med (2018) 15(6):721-733 [Overview of the project] [Problems that the invention aims to solve]

[0008] Wound healing models are used to evaluate the effectiveness of wound treatment methods, but while conventional in vitro model systems can evaluate epithelialization, they cannot evaluate dermal healing at all. In order to reproduce healing, it is desirable to develop a model that reproduces the process in which granulation tissue is produced at the wound site and the wound closes.Therefore, one of the objectives of the present invention is to provide a skin tissue wound healing mimic that produces granulation tissue at the wound site. [Means for solving the problem]

[0009] To solve the above problems, the inventors conducted extensive research and discovered that by setting the collagen concentration in a cell culture gel within a specific range and the cell density in the gel within a specific range, wounds are created in the gel, and these wounds heal through subsequent cell culture in the gel. This led to the completion of the present invention.

[0010] A skin tissue wound healing mimic according to an aspect of the present invention comprises a gel of cells and collagen, wherein the average concentration of collagen in the skin tissue wound healing mimic is 2 % The above 10 % below (20mg / mL or more and 100mg / mL or less) That is the case.

[0011] In the above-mentioned skin tissue wound healing mimicry material, (average concentration of collagen [ % ] × 2) + log(number of cells per unit volume [cells / mL]) However, a score between 9.3 and 29.0 is also acceptable.

[0012] In the above-described skin tissue wound healing mimic, cells may include fibroblasts.

[0013] The above-mentioned skin tissue wound healing mimic material includes multiple regions with different collagen concentrations, and the collagen concentration in at least a portion of the multiple regions is 0.1 % from 0.5 % (1 mg / mL to 5 mg / mL) Therefore, the collagen concentration in at least some other parts of multiple regions is 2 % from 8 % (20 mg / mL to 80 mg / mL)It may be.

[0014] The method for observing a skin tissue wound healing mimic material according to an aspect of the present invention is a skin tissue wound healing mimic material containing cells and a collagen gel, wherein the average concentration of collagen in the skin tissue wound healing mimic material is 2 % or more and 10 % or less (20mg / mL or more and 100mg / mL or less) preparing a skin tissue wound healing mimic material, cutting the skin tissue wound healing mimic material, bringing the cut portions of the skin tissue wound healing mimic material into contact with each other, and observing the healing of the cut portions.

[0015] In the method for observing the skin tissue wound healing mimic material described above, when observing the healing of the cut portions, the presence or degree of fracture when the skin tissue wound healing mimic material is stretched may be observed.

[0016] In the method for observing the skin tissue wound healing mimic material described above, when stretching the skin tissue wound healing mimic material, one side of the cut portion of the skin tissue wound healing mimic material may be gripped and the other side may be pulled.

[0017] In the method for observing the skin tissue wound healing mimic material described above, before observing the healing of the cut portions, the cell medium may be replaced with a calcium-free solution. In the method for observing the skin tissue wound healing mimic material described above, before observing the healing of the cut portions, the calcium in the skin tissue wound healing mimic material may be chelated.

[0018] The screening method for a skin tissue wound treatment agent according to an aspect of the present invention is a skin tissue wound healing mimic material containing cells and a collagen gel, wherein the average concentration of collagen in the skin tissue wound healing mimic material is 2 % or more and 10 <好 % or less (20mg / mL or more and 100mg / mL or less) preparing a skin tissue wound healing mimic material, cutting the skin tissue wound healing mimic material, bringing the cut portions of the skin tissue wound healing mimic material into contact with each other, adding a wound treatment agent to the cut portions, and observing the healing of the cut portions.

[0019] In the method for screening the above-described skin tissue wound treatment agent, when observing the healing between the cut sites, the presence or degree of fracture when the skin tissue wound healing mimic material is stretched may be observed.

[0020] In the method for screening the above-described skin tissue wound treatment agent, when stretching the skin tissue wound healing mimic material, one side may be gripped with respect to the cut site of the skin tissue wound healing mimic material, and the other side may be pulled.

[0021] In the method for screening the above-described skin tissue wound treatment agent, the cell culture medium may be replaced with a calcium-free solution before observing the healing between the cut sites. In the method for screening the above-described skin tissue wound healing mimic material, the calcium in the skin tissue wound healing mimic material may be chelated before observing the healing between the cut sites.

[0022] The method for producing a skin tissue wound healing mimic material according to an aspect of the present invention includes forming a lattice-like skeleton with a collagen solution of a first concentration, filling the gaps of the lattice-like skeleton with a collagen solution of a second concentration containing cells and having a lower concentration than the first concentration, and gelling the collagen solution of the first concentration and the collagen solution of the second concentration to obtain a skin tissue wound healing mimic material. The concentration of collagen in the collagen solution of the first concentration is 2 % 8 or more % hereinafter (20 mg / mL to 80 mg / mL) and the concentration of collagen in the collagen solution of the second concentration is 0.1 % 0.5 or more % hereinafter (1mg / mL or more and 5mg / mL or less) and the average concentration of collagen in the entire skin tissue wound healing mimic material is 2 % 10 or more % hereinafter (20mg / mL or more and 100mg / mL or less) The volume of the collagen solution of the first concentration and the volume of the collagen solution of the second concentration are set so as to satisfy the above conditions.

[0023] In the method for producing the above-described skin tissue wound healing mimic material, In the entire skin tissue wound healing mimic material, (Average concentration of collagen [ % ] × 2) + log(number of cells per unit volume [cells / mL]) However, a collagen solution of a first concentration and a collagen solution of a second concentration may be prepared such that the ratio is between 9.3 and 29.0.

[0024] In the method for producing the above-described skin tissue wound healing mimic, the cells may include fibroblasts.

[0025] In the above-described method for manufacturing a skin tissue wound healing mimic, a lattice-like skeleton may be formed by 3D printing.

[0026] In the above-described method for manufacturing a skin tissue wound healing mimic, a collagen solution of a second concentration may be filled into the gaps of the lattice-like skeleton by 3D printing. [Effects of the Invention]

[0027] According to the present invention, it is possible to provide a skin tissue wound healing mimic that produces tissue mimicking granulation tissue at the wound site. [Brief explanation of the drawing]

[0028] [Figure 1] Figure 1 shows the toolpath used when preparing the skin tissue wound healing mimic material according to Example 9. [Figure 2] Figure 2 shows the structure of the skin tissue wound healing mimic material according to Example 9. [Figure 3] Figure 3 shows a cross-section of the toolpath of the skin tissue wound healing mimic material according to Example 9. [Figure 4] Figure 4 shows the experimental procedures for Examples 1 to 8 and Comparative Examples 1 to 19. [Figure 5] Figure 5 shows the skin tissue wound healing mimicry material according to Example 1. [Figure 6] Figure 6 shows the skin tissue wound healing mimicry material according to Example 1. [Figure 7] Figure 7 shows the skin tissue wound healing mimicry material according to Example 1. [Figure 8] Figure 8 shows the skin tissue wound healing mimicry material related to Comparative Example 1. [Figure 9] Figure 9 shows the skin tissue wound healing mimicry material related to Comparative Example 5. [Figure 10] Figure 10 shows the skin tissue wound healing mimicry material related to Comparative Example 17. [Figure 11] Figure 11 is a graph showing the relationship between the collagen concentration in a skin tissue wound healing mimic, the cell density, and the success or failure of healing. [Figure 12] Figure 12 shows the skin tissue wound healing mimicry material according to Example 9. [Modes for carrying out the invention]

[0029] Embodiments of the present invention are described below. However, these embodiments should not be understood as limiting the present invention. Various alternative embodiments, examples, and operational techniques should become apparent to those skilled in the art from this disclosure. It should be understood that the present invention encompasses various embodiments and the like that are not described herein.

[0030] The skin tissue wound healing mimicry material according to this embodiment comprises a gel of cells and collagen, and the average concentration of collagen in the skin tissue wound healing mimicry material is 2 % The above 10 % The following applies:

[0031] Cells that produce collagen, as well as cells found in the skin and dermis, can be used. Examples of collagen-producing cells include fibroblasts, mesenchymal stem cells, and chondrocytes. Examples of fibroblasts include cutaneous fibroblasts, pulmonary fibroblasts, cardiac fibroblasts, aortic adventitia fibroblasts, uterine fibroblasts, and villous mesenchymal fibroblasts. Examples of cells found in the skin and dermis include keratinocytes, Langerhans cells, melanocytes, mast cells, vascular endothelial cells, pericytes, and immune cells. Examples of cell origins include human, mouse, and rat. The cells are cultured, proliferate, and migrate within the collagen gel.

[0032] Examples of collagen include type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XV collagen, type XVII collagen, and type XVIII collagen. Collagen is preferably fibrous collagen. Collagen is preferably type I collagen or type III collagen.

[0033] The collagen gel is preferably prepared in a way that allows for cell culture. For example, the collagen gel preparation steps, such as drying, swelling, concentration adjustment, and neutralization, are preferably carried out under sterile conditions.

[0034] The average concentration of collagen in the skin tissue wound healing mimic material according to the embodiment is 2 % The above 10 % The following applies. Here, the average collagen concentration is the average collagen concentration in the entire skin tissue wound healing mimic, and the collagen concentration in the skin tissue wound healing mimic is 2 % The above 10 % Even if there are parts outside the following range, the average collagen concentration as a whole is 2 % The above 10 % The following conditions are acceptable. For example, in a skin tissue wound healing mimic, the collagen concentration is 2 % The portion that is less than 10, or the collagen concentration is 10 % Even if there are areas with higher concentrations, the overall average collagen concentration is 2 % The above 10 % The following is acceptable.

[0035] The average concentration of collagen in skin tissue wound healing mimicry materials is 2 % As a result, it is possible to suppress deformation or collapse of the collagen gel in the skin tissue wound healing mimic during cell culture, or during or after wound creation. Furthermore, the average concentration of collagen in the skin tissue wound healing mimic is 10 %The following conditions make it possible to prepare uniformly swollen collagen when swelling freeze-dried collagen during the preparation of skin tissue wound healing mimicry materials.

[0036] A skin tissue wound healing mimic is manufactured, for example, by mixing a collagen solution with cells and gelling the collagen solution containing the cells in a container. The skin tissue wound healing mimic may further include a culture medium for culturing the cells within the collagen gel.

[0037] The skin tissue wound healing mimic may be manufactured using a 3D printer. For example, a lattice-like skeleton may be formed on a substrate with a collagen solution of a first concentration, and the gaps in the lattice-like skeleton may be filled with a collagen solution containing cells, which is a collagen solution of a second concentration lower than the first concentration, and the first and second concentrations of collagen solutions may be gelled. The first concentration of collagen solution may be gelled before filling with the second concentration of collagen solution. Alternatively, the first and second concentrations of collagen solution may be gelled simultaneously.

[0038] When the skin tissue wound healing mimic is manufactured using a 3D printer, the first concentration is, for example, 2 % The above 8 % The following applies: The first concentration is 2 % The above 8 % The following conditions allow for the layering of the skeleton during 3D printing and suppression of nozzle clogging during 3D printing. Furthermore, the second concentration is, for example, 0.1 % 0.5 % The following applies: The second concentration is 0.1 % 0.5 % The following conditions allow for the use of general scaffolding containing collagen.

[0039] In skin tissue wound healing mimicry materials, (Average concentration of collagen [ % ] × 2) + log(number of cells per unit volume [cells / mL]) However, the collagen concentration and cell density may be set so that the ratio is between 9.3 and 29.0. This allows the severed area of ​​the skin tissue wound healing mimic to heal.

[0040] The skin tissue wound healing mimic is placed, for example, in a container that completely encloses the skin tissue wound healing mimic. By completely enclosing the skin tissue wound healing mimic in the container, it is possible to suppress the migration of the wound while cells are being cultured within the skin tissue wound healing mimic. The container is, for example, sterilized.

[0041] A method for observing a skin tissue wound healing mimic according to the embodiment includes preparing the skin tissue wound healing mimic according to the embodiment described above, cutting the skin tissue wound healing mimic, bringing the cut portions of the skin tissue wound healing mimic into contact with each other, and observing the healing of the cut portions.

[0042] Cells are cultured within the skin tissue wound healing mimic by adding a culture medium to it. Cutting may be performed with a scalpel. The skin tissue wound healing mimic may be partially cut or divided into two.

[0043] For example, after a period of time has elapsed since cutting the skin tissue wound healing mimic, the skin tissue wound healing mimic is stretched to observe whether or not there is a rupture and to what extent. When stretching the skin tissue wound healing mimic, one side of the skin tissue wound healing mimic may be grasped relative to the cut site, and the other side may be pulled.

[0044] If the skin tissue wound healing imitation material does not rupture when stretched, it can be evaluated that the severed areas have healed. If there is partial rupture but the degree of rupture is low, it can be evaluated that the degree of healing between the severed areas is high. If there is partial rupture and the degree of rupture is high, it can be evaluated that the degree of healing between the severed areas is low. If there is a complete rupture, it can be evaluated that the severed areas have not healed.

[0045] Cells adhere to each other through cadherins, glycoproteins present on the cell surface. Cadherins adhere to cells in a calcium-dependent manner. To confirm whether the skin tissue wound healing mimic maintains healing under conditions where intercellular bonding by cadherins does not occur, the skin tissue wound healing mimic may be immersed in a calcium-free solution and the cell culture medium replaced with a calcium-free solution before observing the healing of the cut sites. Calcium may also be chelated. If the skin tissue wound healing mimic maintains healing under conditions where intercellular bonding by cadherins does not occur, it is possible to evaluate that the wound has been repaired by a collagen matrix produced by the cells. Wound repair by the collagen matrix mimics the formation of granulation tissue.

[0046] In the above method for observing skin tissue wound healing mimics, a wound healing agent may be added to the ruptured area to screen for effectiveness. If the skin tissue wound healing mimic to which the wound healing agent has been added does not rupture when stretched, the wound healing agent can be evaluated as effective. If there is partial rupture but the degree of rupture is low, the wound healing agent can be evaluated as somewhat effective. If there is partial rupture and the degree of rupture is high, the effectiveness of the wound healing agent can be evaluated as low. If there is complete rupture, the wound healing agent can be evaluated as ineffective.

[0047] (Examples) Examples of the present invention are described below. However, it goes without saying that the present invention is not limited to the following examples.

[0048] (Preparation of collagen) All collagen preparations were performed in a clean bench, and all reagents and equipment used were sterilized. A commercially available 0.5% collagen (mainly type I) solution was filled into freeze-drying containers under sterile conditions and frozen at -80°C. The frozen collagen samples were dried in a freeze-drying oven. After drying, the weight of the collagen samples was measured, and hydrochloric acid was added to the dried collagen samples under sterile conditions to achieve a collagen concentration of 10%. Subsequently, the dried collagen samples were swollen at 4°C, and hydrochloric acid, 10-fold concentrated saline phosphate, phenol red, and 1 mol / L sodium hydroxide aqueous solution were added to the collagen samples and mixed thoroughly to neutralize them. The collagen concentration in the collagen samples was controlled by controlling the amount of hydrochloric acid mixed during neutralization.

[0049] (Preparation of skin tissue wound healing mimicry materials according to Examples 1 to 8 and Comparative Examples 1 to 19) Human fibroblasts (passage 6) were grown in culture medium (Dulbecco's Modified Eagle Medium, containing 10% fetal bovine serum and 1% penicillin / streptomycin). A cell suspension was then prepared using a standard cell detachment procedure with trypsin / EDTA aqueous solution. The cell suspension was mixed with a neutralized collagen solution, and 150 μL of the collagen solution containing the cells was added to a 48-well dish. After confirming that the solution was uniformly spread throughout the wells without gaps, the collagen solution was gelled at 37°C for 1 hour to prepare skin tissue wound healing mimics according to Examples 1 to 8 and Comparative Examples 1 to 19. The collagen concentration and cell density in each skin tissue wound healing mimic are shown in Table 1. [Table 1]

[0050] (Preparation of a skin tissue wound healing imitation material according to Example 9) A 4% collagen solution was filled into a 3D printer cartridge and fitted with a 22G nozzle. A 0.4% collagen solution containing cells was also filled into a 3D printer cartridge and fitted with an 18G nozzle. Each cartridge was then mounted on the 3D printer head. Following the toolpath shown in Figure 1, a lattice-like framework made of 4% collagen was fabricated using 3D printing (4% collagen: extrusion pressure 135kPa, 0.4% collagen + cells: extrusion pressure 36kPa, head temperature 4°C, stage temperature 37°C). The framework was then filled with 0.4% collagen containing cells to create the structure shown in Figure 2. Figure 3 shows cross-sectional views of the toolpaths in each layer of the fabricated structure. Calculating the gelled composition from these toolpaths, the average collagen concentration was 3.6%, and the average cell density was 2 × 10⁻⁶. 5 The concentration was cells / mL. After 3D printing the structure, the solution was gelled at 37°C for 1 hour to produce the skin tissue wound healing mimicry material according to Example 9.

[0051] (Experimental procedures for Examples 1 to 8 and Comparative Examples 1 to 19) As shown in Figure 4, 300 μL of culture medium was added to the skin tissue wound healing mimic in the well, and three-dimensional cell culture (37°C, 5% CO2 atmosphere) was started in a collagen gel. The next day, the skin tissue wound healing mimic was cut in half using a sterile scalpel. After confirming that the cut surfaces of the cut skin tissue wound healing mimic were in contact, the cells were further cultured in three dimensions. During cell culture, the culture medium was changed every 2-3 days. After cutting the skin tissue wound healing mimic, the cells were cultured in three dimensions for 10 days.

[0052] (Method for confirming fusion) Ten days after culturing the cells, the skin tissue wound healing mimic was grasped and lifted with tweezers to confirm healing. The wound was also observed under a microscope.

[0053] (Confirmation of extracellular matrix) On day 14 of culture, the skin tissue wound imitation material was grasped with tweezers and lifted, and the healing of the cut surface was confirmed. In addition, the inside of the skin tissue wound imitation material was observed to confirm the collagen produced at the wound site. Frozen sections with a thickness of 30 μm were prepared from the cultured skin tissue wound imitation material, and the frozen sections were stained with hematoxylin and eosin and observed.

[0054] (Evaluation results of skin tissue wound healing mimicry materials related to Examples 1 to 8) For the skin tissue wound healing mimics described in Examples 1 to 8, after cutting, the skin tissue wound healing mimics were moved in a well on the 10th day to confirm healing. As shown in Figure 5a, the adhesion of the wound was visually confirmed. Furthermore, when the skin tissue wound healing mimic was moved with the tip of tweezers, it was confirmed that the two cut gels were adhering to each other. Microscopic observation at 100x magnification also confirmed, as shown in Figure 5b, that the wound had healed and the two skin tissue wound healing mimics were adhering to each other. These results demonstrate that wound healing occurs when cells are cultured in contact with the wound area of ​​the skin tissue wound healing mimic.

[0055] Furthermore, to confirm the maintenance of healing under conditions where intercellular bonding by cadherin does not occur, the skin tissue wound healing mimic material according to Example 1 was immersed in 5 mmol / L EDTA / phosphate saline on the 14th day after culturing the cells. As shown in Figure 6a, the healing was confirmed again on the 3rd day of immersion in phosphate saline, and as shown in Figure 6b, it was confirmed that the wound did not rupture even when picked up with tweezers. The fact that the wound did not rupture despite the EDTA / phosphate saline chelating the calcium necessary for intercellular adhesion and eliminating the calcium necessary for intercellular adhesion indicates that not only intercellular bonding but also bonding via the extracellular matrix occurs in wound healing.

[0056] Furthermore, to confirm the extracellular matrix produced during repair, frozen sections of the skin tissue wound healing mimic material according to Example 1 were prepared with a thickness of 30 μm and stained with hematoxylin and eosin. As a result, as shown in Figure 7, it was confirmed that the wound in the area indicated by the arrow was filled with collagen. Since the skin tissue wound healing mimic material according to Example 1 did not involve collagen transplantation into the wound as performed in conventional techniques, this demonstrates that the wound was repaired by a collagen matrix produced from cells. This indicates that tissue mimicking granulation tissue was produced at the wound site.

[0057] (Evaluation results of skin tissue wound healing mimicry materials related to Comparative Examples 1 to 19) Regarding the skin tissue wound healing mimics of Comparative Examples 1 to 19, when the skin tissue wound healing mimics were moved in the well 10 days after cutting, no healing could be confirmed. In the skin tissue wound healing mimics of Comparative Examples 1 to 4 and Comparative Examples 6 to 9, as shown in Figure 8a, healing could not be visually confirmed. Furthermore, as shown in Figure 8b, when observed under a microscope at 100x magnification, gaps were observed in the wound. As shown in Figure 9, in the skin tissue wound healing mimics of Comparative Examples 5, 10, 11, 15, and 16, shrinkage occurred and the wounds dehisced. As shown in Figure 10, in the skin tissue wound healing mimics of Comparative Examples 12 to 14 and 17 to 19, the gel disintegrated.

[0058] (Relationship between collagen concentration and fusion) Figure 11 shows the relationship between the collagen concentration, cell density, and the success or failure of healing in the skin tissue wound healing mimicry materials for Examples 1 to 6 and Comparative Examples 1 to 19. From these results, the collagen concentration and cell density required for the healing of cleaved collagen gel when fibroblasts are cultured three-dimensionally in a collagen gel have been clarified. The region where fusion occurs in the graph of Figure 11 can be calculated from the straight line connecting Examples 8 and 6. Collagen concentration ≥ 2%, (Collagen concentration [%] × 2) + log(cells per unit volume [cells / mL]) ≥ 9.301 The conditions are met.

[0059] The upper limit of collagen concentration that can be prepared by freeze-drying is 10%, and the upper limit of cell density concentration that can be prepared as a collagen gel is 1 × 10⁻⁶ 9 The value is cells / mL. Therefore, the conditions under which healing occurs in a skin tissue wound healing mimic is: 29 ≥ (Collagen concentration [%] × 2) + log(Cell count per unit volume [cells / mL]) ≥ 9.301 The collagen concentration and cell density were calculated to satisfy the given conditions.

[0060] (Evaluation results of the skin tissue wound healing mimicry material according to Example 9) As shown in Figure 12a, for the skin tissue wound healing mimic material according to Example 9, healing was confirmed on the 10th day after cutting by moving the gel in the well. As shown in Figure 12b, adhesion of the wound was visually confirmed, and the adhesion of the two cut gels was confirmed when the gel was moved with the tip of tweezers. The overall composition of the skin tissue wound healing mimic material according to Example 9 was an average collagen concentration of 3.6% and an average cell density of 2 × 10⁻⁶ 5 The cell density is cells / mL, which falls within the range of collagen composition for "wound healing" shown in the graph in Figure 11. Therefore, it was shown that the collagen concentration and cell density conditions for wound healing shown in Figure 11 can be applied to gels with a combination of multiple collagen concentrations.

Claims

1. A skin tissue wound healing mimic comprising collagen-producing cells and a collagen gel, The average concentration of collagen in the skin tissue wound healing mimic is 2% or more and 10% or less (20 mg / mL or more and 100 mg / mL or less), (Average concentration of collagen [%] × 2) + log(Number of cells per unit volume [cells / mL]) However, it is between 9.3 and 29.

0. A material that mimics the healing of skin tissue wounds.

2. The skin tissue wound healing mimicry material according to claim 1, wherein the cells include fibroblasts.

3. The skin tissue wound healing mimic according to claim 1, wherein the skin tissue wound healing mimic comprises a plurality of regions with different collagen concentrations, wherein the collagen concentration in at least a portion of the plurality of regions is 0.1% to 0.5% (1 mg / mL to 5 mg / mL), and the collagen concentration in at least another portion of the plurality of regions is 2% to 8% (20 mg / mL to 80 mg / mL).

4. A skin tissue wound healing mimic comprising collagen-producing cells and a collagen gel, wherein the average concentration of collagen in the skin tissue wound healing mimic is 2% or more and 10% or less (20 mg / mL or more and 100 mg / mL or less), Cutting the aforementioned skin tissue wound healing mimicry material, Bringing the cut ends of the aforementioned skin tissue wound healing mimic material into contact with each other, Observe the fusion of the aforementioned cut parts, Includes, (Average concentration of collagen [%] × 2) + log(Number of cells per unit volume [cells / mL]) However, it is between 9.3 and 29.

0. Method for observing skin tissue wound healing mimicry materials.

5. A method for observing a skin tissue wound healing mimic, according to claim 4, wherein, in observing the healing of the aforementioned severed areas, the presence or degree of rupture when the skin tissue wound healing mimic is stretched is observed.

6. The method for observing a skin tissue wound healing mimic, according to claim 5, wherein when stretching the skin tissue wound healing mimic, one side of the skin tissue wound healing mimic is grasped with respect to the cut portion of the skin tissue wound healing mimic and the other side is pulled.

7. The method for observing a skin tissue wound healing mimic according to claim 4, wherein, before observing the fusion of the aforementioned severed parts, the culture medium of the cells is replaced with a calcium-free solution.

8. A skin tissue wound healing mimic comprising collagen-producing cells and a collagen gel, wherein the average concentration of collagen in the skin tissue wound healing mimic is 2% or more and 10% or less (20 mg / mL or more and 100 mg / mL or less), Cutting the aforementioned skin tissue wound healing mimicry material, Bringing the cut ends of the aforementioned skin tissue wound healing mimic material into contact with each other, Adding a wound healing agent to the aforementioned cut site, Observe the fusion of the aforementioned cut parts, Includes, (Average concentration of collagen [%] × 2) + log(Number of cells per unit volume [cells / mL]) However, it is between 9.3 and 29.

0. A screening method for skin tissue wound treatment agents.

9. A screening method for a skin tissue wound treatment agent according to claim 8, wherein, in observing the healing of the aforementioned severed areas, the presence or degree of rupture when the skin tissue wound healing mimic material is stretched is observed.

10. A method for screening a skin tissue wound treatment agent according to claim 9, wherein when stretching the skin tissue wound healing mimic, one side of the skin tissue wound healing mimic is grasped with respect to the cut portion and the other side is pulled.

11. A method for screening a skin tissue wound treatment agent according to claim 8, wherein, before observing the fusion of the aforementioned severed areas, the culture medium of the cells is replaced with a calcium-free solution.

12. Forming a lattice-like skeleton with a collagen solution of the first concentration, The gaps in the aforementioned lattice-like skeleton are filled with a collagen solution containing cells and having a second concentration lower than the first concentration. To obtain a skin tissue wound healing mimic by gelling the collagen solution of the first concentration and the collagen solution of the second concentration, A method for producing a skin tissue wound healing mimic, which includes: The collagen concentration in the collagen solution of the first concentration is 2% or more and 8% or less (20 mg / mL or more and 80 mg / mL or less), The collagen concentration in the second concentration collagen solution is 0.1% or more and 0.5% or less (1 mg / mL or more and 5 mg / mL or less), The volumes of the collagen solution of the first concentration and the collagen solution of the second concentration are set such that the average concentration of collagen in the entire skin tissue wound healing mimic material is 2% or more and 10% or less (20 mg / mL or more and 100 mg / mL or less). In the entire aforementioned skin tissue wound healing mimicry material, (Average concentration of collagen [%] × 2) + log(Number of cells per unit volume [cells / mL]) However, the collagen solution of the first concentration and the collagen solution of the second concentration are prepared so that the ratio is between 9.3 and 29.

0. A method for manufacturing a skin tissue wound healing mimic.

13. A method for producing a skin tissue wound healing mimic according to claim 12, wherein the cells include fibroblasts.

14. A method for producing a skin tissue wound healing mimic material according to claim 12, wherein the lattice-like skeleton is formed by 3D printing.

15. A method for producing a skin tissue wound healing mimic according to claim 12, wherein the gaps in the lattice-like skeleton are filled with the collagen solution of the second concentration by 3D printing.

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